CN116429870A - Method for eliminating imaging mass spectrum flow type sensitivity difference - Google Patents
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Abstract
The invention provides a signal calibration method of an imaging mass spectrometry flow type, which carries out positive and negative linear regression on a sample signal through a standard signal: firstly, selecting a sample as a standard sample, establishing a first standard curve and a first regression model according to the standard on a glass slide of the sample, then establishing a second standard curve and a second regression model according to the standard on the glass slide of the sample to be calibrated, then adopting a first resolution to scan the sample to be calibrated, performing log processing on an obtained original signal value, inputting the second regression model to obtain the actual metal content of each pixel of the sample to be calibrated, and finally converting the actual metal content of each pixel into a calibration signal value through the first regression model to finish data calibration, thereby eliminating the influence caused by instrument sensitivity fluctuation.
Description
Technical Field
The invention relates to a multichannel imaging technology, in particular to a signal calibration method of an imaging mass spectrum flow type.
Background
Imaging mass spectrometry (Imaging Mass Cytometry) is a platform of tissue multichannel imaging technology. The method comprises the steps of marking a tissue sample by using a metal tag antibody to form a tissue section, scanning and sampling point by laser, and then sending the tissue section into an inductively coupled plasma mass spectrometry (ICP-mass) host for elemental analysis, so that distribution information of the metal tag in a detected area is obtained, and images of dozens of channels in the same visual field are reconstructed. Compared with other fluorescence-based tissue imaging technologies, the imaging mass spectrometry has the advantages of multiple channels, no cross color, no interference from tissue background fluorescence and the like, so that the imaging mass spectrometry has an important role in the research of tissue microenvironments of tumors, type I diabetes and some infectious diseases.
However, due to the comprehensive influence of factors such as the environment and oxidization of components related to the sampling cone, the sensitivity of the imaging mass spectrum flow type can fluctuate, the strength of detected signals is directly influenced, and further, the consistency of data and the follow-up raw data analysis can be influenced to a certain extent. In order to obtain more accurate data analysis results, it is important how to eliminate the influence caused by the fluctuation of the sensitivity of the instrument.
Disclosure of Invention
In order to eliminate the influence of instrument sensitivity fluctuation on a sample signal, the invention provides an imaging mass spectrometry flow type signal calibration method, which calibrates the sample signal through a standard signal, and comprises the following steps:
selecting one sample as a standard sample, and establishing a first standard curve according to the standard sample on the sample;
establishing a first regression model according to the first standard curve, wherein the first regression model is used for converting the actual metal content into a signal value;
establishing a second standard curve according to the standard substance on the sample to be calibrated; establishing a second regression model according to the second standard curve, wherein the second regression model is used for converting the original signal value subjected to log processing into actual metal content;
scanning a sample to be calibrated, and calculating the actual metal content of each pixel of the sample to be calibrated according to the second regression model;
and converting the calculated actual metal content of each pixel into a calibration signal value through a first regression model, and completing the calibration of data.
Further, the establishing of the first standard curve and/or the second standard curve includes:
defining a scanning area for a standard on a sample slide; and
scanning the standard region of interest to establish a standard curve, wherein the sample slide includes at least three standards thereon.
Further, the resolution employed to scan the region of interest of the standard is lower than the resolution of scanning the sample to be calibrated.
Further, the method also includes eliminating sensitivity differences inside the sample region and between the sample region and the standard region based on the change in signal ratio of argon dimer, or xenon, or iodine elements of the sample region and the standard region.
Further, the standard comprises one or more halides and/or soluble salts comprising a lanthanide metal, and the atomic weight of the standard covers the range 139 to 176 lanthanides.
Further, the standard includes cerium chloride, samarium nitrate, holmium chloride, and lutetium chloride.
Further, the standard is set on a slide of a sample by:
forming a plurality of standard dilutions of different concentrations;
mixing the multiple standard substance dilutions with trypan blue with specified concentration respectively to obtain multiple working solutions;
heating a portion of the slide; and
after the designated time period, the designated amount of the various working fluids are respectively dispensed to the heated portions of the slide.
Further, the forming of the standard diluent comprises:
the metal salt is diluted with dilute hydrochloric acid of the specified concentration.
Further, the concentration of the diluted hydrochloric acid is 0.01M, and the concentration of the standard substance diluent is 10 -4 M to 10 -8 M.
Further, the number of the standard substance dilutions is three, and the concentrations of the three standard substance dilutions are respectively as follows: 10 - 6 M、10 -7 M and 10 -8 M。
Further, the concentration of trypan blue is 0.5%, which is mixed with the standard diluent 1:1.
Further, heating the portion of the slide includes:
and heating the part, needing to be provided with the standard substance, on the slide by the heating module.
Further, the heating range of the heating module is 40-70 ℃.
The invention provides a signal calibration method of an imaging mass spectrum flow type, which takes a standard solution point containing a series of lanthanide metals as a standard substance on one side of a sample. The instrument scans the standard area before detecting the sample, and then scans the sample. Because the metal content in the standard substance area is fixed, the signal intensity of the standard substance area can change along with the sensitivity of the instrument, so that the signal of a sample can be calibrated by utilizing the signal of the standard substance, and the influence caused by the fluctuation of the sensitivity of the instrument is eliminated. The signal calibration method further carries out positive and negative linear regression on the sample to be calibrated through the standard sample, and further eliminates the influence caused by the sensitivity fluctuation of the instrument.
Drawings
To further clarify the above and other advantages and features of embodiments of the present invention, a more particular description of embodiments of the invention will be rendered by reference to the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. In the drawings, for clarity, the same or corresponding parts will be designated by the same or similar reference numerals.
FIG. 1 is a flow chart of a method of signal calibration of an imaging mass spectrometry according to an embodiment of the present invention;
FIG. 2 shows a schematic view of localized heating of a slide in accordance with one embodiment of the invention;
FIGS. 3a, 3b show schematic views of images scanned at different sensitivities using an imaging mass spectrometry flow;
FIGS. 3c and 3d are schematic diagrams of signal calibration of FIGS. 3a and 3b, respectively, using an imaging mass spectrometry method according to one embodiment of the present invention;
FIG. 3e shows a schematic diagram of the statistics of FIGS. 3a to 3 d;
FIGS. 4a and 4b are schematic diagrams of an imaging mass spectrometry method for calibrating an image of a tissue region according to an embodiment of the present invention;
FIGS. 5a and 5b are schematic diagrams of a signal calibration method of an imaging mass spectrometry method for calibrating an image of a further tissue region according to an embodiment of the present invention;
FIGS. 5c and 5d are schematic diagrams showing the tsne dimension reduction analysis corresponding to FIGS. 5a and 5b, respectively;
FIG. 6a shows sample area argon dimer signal variation in one embodiment of the present invention;
FIG. 6b shows the adj.factor for each row calculated from the argon dimer signal variation case shown in FIG. 6 a; and
fig. 6c and 6d show schematic signal distribution diagrams of HistoneH3 before and after calibration by the argon dimer signal calibration method according to an embodiment of the present invention.
Detailed Description
In the following description, the present invention is described with reference to various embodiments. One skilled in the relevant art will recognize, however, that the embodiments may be practiced without one or more of the specific details, or with other alternative and/or additional methods, materials, or components. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention. Similarly, for purposes of explanation, specific numbers, materials and configurations are set forth in order to provide a thorough understanding of the embodiments of the invention. However, the invention is not limited to these specific details. Furthermore, it should be understood that the embodiments shown in the drawings are illustrative representations and are not necessarily drawn to scale.
Reference throughout this specification to "one embodiment" or "the embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment.
It should be noted that the embodiments of the present invention describe the steps of the method in a specific order, however, this is merely for the purpose of illustrating the specific embodiments, and not for limiting the order of the steps. In contrast, in different embodiments of the present invention, the sequence of each step may be adjusted according to the adjustment of the actual requirement.
Aiming at the problem that the existing imaging mass spectrometry is easy to be influenced by instrument sensitivity change when the tissue multichannel imaging is carried out, the invention provides an imaging mass spectrometry signal calibration method, which uses signals of a standard to calibrate signals of a sample and eliminates the influence caused by instrument sensitivity fluctuation.
In the present embodiment, the standard is a standard solution containing a series of lanthanide metals, with the spot on one side of the sample, and the instrument scans the standard area at a lower resolution, e.g., 5 to 10um, before scanning the sample at a normal higher resolution, e.g., 1 um. Because the metal content in the standard substance area is fixed and the signal intensity of the standard substance area changes along with the sensitivity of the instrument, the signal of the sample can be calibrated by utilizing the signal of the standard substance, and the influence caused by the fluctuation of the sensitivity of the instrument is eliminated. In one embodiment of the invention, the template of the standard sample is set to: the laser energy is set to 2, the x-step and y-step are set to 10, the number of lanthanide metal channels between 139 and 176 is selected, and before scanning, a scan area needs to be defined for each standard area, ensuring that the entire standard area is scoped, and in one embodiment of the invention, scanning the ROI area of a single standard takes about 5 minutes.
In one embodiment of the invention, the standard is a diluted metal salt, including, for example, lanthanide metals such as cerium Ce, samarium Sm, holmium Ho, lutetium Lu, and the like, other lanthanide metals similar in chemical nature thereto, or halide, nitrate, acetate, or other soluble salt forms comprising lanthanum, praseodymium, neodymium, promethium, europium, gadolinium, terbium, dysprosium, erbium, thulium, ytterbium, and the like. In one embodiment of the invention, the atomic weight of the metal salt should cover the range 139 to 176 lanthanoid, table 1 gives the isotopic abundance of the relevant element, and the metal salt can be selected according to table 1.
TABLE 1
In one embodiment of the invention, cerium chloride, samarium nitrate, holmium chloride and lutetium chloride are selected as standard substances, and after all metal salts are accurately weighed, the metal salts are diluted into a plurality of standard substance dilutions with different concentrations by adopting 0.01M dilute hydrochloric acid, and the standard substance dilutions are stored for later use. In one embodiment of the invention, the concentration of the standard diluent is 10 -4 M to 10 -8 M. In one embodiment of the invention, three standards are used and the concentration of each standard diluent is 10 -6 M、10 -7 M and 10 -8 M。
The standard diluent is further mixed with trypan blue with a specified concentration according to a preset proportion to obtain the required standard solution. In one embodiment of the present invention, 0.5% trypan blue and standard dilutions of different concentrations were used 1:1, and obtaining a standard solution.
In one embodiment of the invention, the standard solution is spotted onto the sample side, first requiring preheating of the slide of the sample section. Fig. 2 shows a schematic view of localized heating of a slide according to one embodiment of the invention. As shown in fig. 2, in one embodiment of the present invention, a portion of a slide where a standard substance needs to be disposed is heated by a heating module, specifically, the heating module is preheated to a temperature ranging from 40 degrees celsius to 70 degrees celsius, preferably to 60 degrees celsius, then a support with a same height is disposed on one side of the heating module, so that one end of a sample slice, that is, a portion where the standard substance needs to be disposed, is placed on the heating module to implement local heating, the other end of the sample slice is placed on the support to keep the level, and after a specified period of heating, standard substance solutions with different concentrations are sequentially sucked and respectively disposed on the slide, and in one embodiment of the present invention, the specified period of time is 1 minute. In yet another embodiment of the invention, the standard solution is allowed to air dry in about 20 seconds to form a circular area of about 1.3mm diameter at 0.3ul spot per concentration on the slide. In order to eliminate the sensitivity difference between the sample region and the standard region, the influence of signal drift (signal drift) may be eliminated according to the change in the signal ratio of the argon dimer, xenon, or iodine element of the sample region and the standard region.
On the basis of setting the standard, the invention further adopts positive and negative linear regression to realize signal calibration, namely firstly converting a scanned signal into metal content through one regression Model, and then converting the metal content into a calibration signal through the other regression Model.
The embodiments of the present invention will be further described with reference to the drawings.
Fig. 1 shows a flow chart of a method for calibrating signals of an imaging mass spectrometry according to an embodiment of the invention. As shown in fig. 1, a signal calibration method of an imaging mass spectrometry includes:
first, in step 101, a first regression model is built. In one embodiment of the invention, the first regression Model R is built from standard samples. Specifically, the establishing of the first regression Model R includes:
firstly, selecting a sample as a standard sample, carrying out antibody and Ir staining on the standard sample, and air-drying the slice;
next, the method as described above is sampled, and a plurality of standard substances are arranged on one side of the standard sample; and
finally, a first standard curve is established according to the standard substance, and a first regression Model R which is converted from the actual metal content into a signal value (count) is established;
next, at step 102, a second regression model is built. In one embodiment of the invention, the second regression Model F is built from the sample to be calibrated. Specifically, the establishing of the second regression Model F includes:
firstly, carrying out antibody and Ir staining on the sample to be calibrated, and air-drying the slice;
next, sampling the method as described above, and setting a plurality of standard substances on one side of the sample to be calibrated; and
finally, a second standard curve is established according to the standard substance, and a second regression Model F is established, wherein the second regression Model F is converted from an original signal value count after log processing, namely log taking operation, to actual metal content;
next, in step 103, the original signal value is acquired. Scanning a sample to be calibrated to obtain an original signal value;
next, at step 104, the actual metal content is calculated. Log processing is carried out on the original signal value, the original signal value is input into the second regression model, and then the actual metal content of each pixel of the sample to be calibrated is calculated; in one embodiment of the present invention, the sensitivity difference inside the sample area and between the sample area and the standard area is also eliminated before the calculation, specifically, the adj.factor of each line of the sample, that is, the ratio of the signal median of the argon dimer, xenon, or iodine element of the line to the signal median of the argon dimer, xenon, or iodine element of the standard area is calculated first, and then the signal value of each channel is divided by the adj.factor line by line to eliminate the influence caused by signal drift (signal drift). FIG. 6a shows sample area argon dimer signal variation in one embodiment of the present invention, and FIG. 6b shows the adj.factor for each row calculated from the argon dimer signal variation shown in FIG. 6 a; and FIGS. 6c and 6d are schematic diagrams showing the signal distribution of Histone H3 before and after calibration by the argon dimer signal calibration method according to one embodiment of the present invention, respectively, as shown in the figures, before calibration, at the position of argon dimer signal shear, i.e. the position indicated by the arrow, obvious signal intensity changes can be seen, and the image signal distribution becomes uniform after calibration, wherein Histone H3 is a nucleosome protein distributed in the nucleus; and
finally, in step 105, a calibration signal value is calculated. And converting the calculated actual metal content of each pixel into a calibration signal value through a first regression model, wherein the calibration signal value is equivalent to a value detected under the condition of adopting the sensitivity which is completely the same as that of the standard sample, thereby completing the standardization of data.
In order to verify the effect of the signal calibration method provided by the invention, different images are calibrated by adopting the signal calibration method.
Figures 3a, 3b show schematic views of images obtained by scanning with an imaging mass spectrometer at different sensitivities, respectively. Wherein, fig. 3a is an image scanned at high sensitivity, and fig. 3b is an image scanned at low sensitivity, it can be seen that there is a significant signal difference in the obtained images at different sensitivities. Fig. 3c and 3d are schematic diagrams respectively showing the calibration of fig. 3a and 3b by an imaging mass spectrometry signal calibration method according to an embodiment of the present invention, and it can be seen that the monitoring data under two sensitivities are substantially the same after signal calibration. To visually display the difference, fig. 3e further shows the statistical data of fig. 3a to 3d, wherein darker colored columns show the statistical data of fig. 3a and 3b, and lighter colored columns show the statistical data of fig. 3c and 3 d. Specifically, in fig. 3e, the content values of Ce, sm, lu obtained from the image statistics are respectively displayed in three regions on the left, two columns on the left side in each region are shown as metal content values corresponding to the acquired image in fig. 3a, 3c, i.e., in high sensitivity, and two columns on the right side are shown as metal content values corresponding to the acquired image in fig. 3b, 3d, i.e., in low sensitivity.
Fig. 4a and 4b are schematic diagrams respectively showing an image of a certain tissue region (tonsil of a human body) before and after calibration by using an imaging mass spectrometry signal calibration method according to an embodiment of the present invention, wherein the upper part of fig. 4a is scanned with high sensitivity, and the lower part is scanned with low sensitivity, so that the lower part is obviously weaker, and after signal calibration, the upper part and the lower part are not obviously distinguished, as shown in fig. 4b, so that the comparability of tissue pictures is significantly improved.
Fig. 5a and 5b show schematic diagrams before and after calibrating a further tissue region by using an imaging mass spectrometry signal calibration method according to an embodiment of the present invention, and fig. 5c and 5d show schematic diagrams of tsne dimension reduction analysis corresponding to fig. 5a and 5b, respectively. Wherein the upper part of FIG. 5a is scanned at low sensitivity and the lower part is scanned at high sensitivity, it can be seen that there is a significant difference in the signals of the two regions, which is evident from the ctsne dimension reduction analysis of FIG. 5, where the cells of the upper low sensitivity region are concentrated, indicating a more pronounced phenotypic difference in the cells of the other regions. After signal calibration, as shown in fig. 5b and 5d, the cell distribution in the region is fused into a large group, the influence of sensitivity is basically eliminated, and the influence of sensitivity difference on the analysis result of the biological information is obviously reduced.
While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. It will be apparent to those skilled in the relevant art that various combinations, modifications, and variations can be made therein without departing from the spirit and scope of the invention. Thus, the breadth and scope of the present invention as disclosed herein should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.
Claims (10)
1. A method of eliminating imaging mass flow sensitivity differences comprising the steps of:
calculating the signal median of argon dimer, xenon or iodine elements of the sample line by line to obtain a first numerical value;
calculating the median of the signals of the argon dimer or the xenon or the iodine element in the standard substance area on the sample line by line to obtain a second numerical value;
calculating the ratio of the first value to the second value row by row; and
dividing the signal value of each channel row by the ratio to eliminate the sensitivity difference of the signal value of each channel.
2. The method as recited in claim 1, further comprising:
scanning a sample to be measured to obtain an original signal value;
log processing is carried out on the signal value after the sensitivity difference is eliminated, and a second regression model is input to obtain the actual metal content of each pixel of the sample to be measured; and
the actual metal content of each pixel is converted into a measured signal value by a first regression model.
3. The method of claim 2, wherein the first regression model is built according to the steps of:
selecting one sample as a standard sample, carrying out antibody and Ir staining on the standard sample, and air-drying the slice;
setting a plurality of standard substances on one side of the standard sample; and
and establishing a first standard curve according to the standard substance, and establishing a first regression model converted from the actual metal content to a signal value.
4. The method of claim 2, wherein the second regression model is built according to the steps of:
carrying out antibody and Ir staining on the sample to be measured, and air-drying and slicing;
setting a plurality of standard substances on one side of the sample to be measured; and
and establishing a second standard curve according to the standard substance, and establishing a second regression model which is converted from the original signal value after the logarithmic operation into the actual metal content.
5. The method of claim 3 or 4, wherein the standard comprises one or more halides and/or soluble salts comprising a lanthanide metal, and the atomic weight of the standard covers the range 139 to 176 lanthanides.
6. The method of claim 3 or 4, wherein the standard is disposed on a slide of the sample by:
diluting the metal salt by adopting dilute hydrochloric acid with specified concentration to form a plurality of standard substance diluents with different concentrations;
mixing the multiple standard substance dilutions with trypan blue with specified concentration respectively to obtain multiple working solutions;
heating a portion of the slide; and
after the designated time period, the designated amount of the various working fluids are respectively dispensed to the heated portions of the slide.
7. The method of claim 6, wherein the dilute hydrochloric acid has a concentration of 0.01M and the standard diluent has a concentration of 10 -4 M to 10 -8 M.
8. The method of claim 6, wherein the concentration of trypan blue is 0.5% mixed with the standard diluent 1:1.
9. The method of claim 6, wherein heating the portion of the slide comprises:
and heating the part, needing to be provided with the standard substance, on the slide by the heating module.
10. The method of claim 9, wherein the heating module heats in a range of 40 degrees celsius to 70 degrees celsius.
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| CN112683986B (en) * | 2021-03-18 | 2021-06-15 | 裕菁科技(上海)有限公司 | Natural isotope calibration curve method for quantifying target analyte in sample |
| CN115015370A (en) * | 2022-06-29 | 2022-09-06 | 中国食品药品检定研究院 | MALDI mass spectrum imaging signal correction method |
| CN116429870A (en) * | 2022-09-30 | 2023-07-14 | 上海立迪生物技术股份有限公司 | Method for eliminating imaging mass spectrum flow type sensitivity difference |
-
2022
- 2022-09-30 CN CN202310515944.1A patent/CN116429870A/en active Pending
- 2022-09-30 CN CN202211205571.XA patent/CN115452929B/en active Active
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2023
- 2023-08-11 WO PCT/CN2023/112512 patent/WO2024066762A1/en unknown
- 2023-08-15 WO PCT/CN2023/113007 patent/WO2024066773A1/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024066762A1 (en) * | 2022-09-30 | 2024-04-04 | 上海立迪生物技术股份有限公司 | Signal calibration method for imaging mass cytometry |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115452929B (en) | 2023-04-21 |
| WO2024066773A1 (en) | 2024-04-04 |
| WO2024066762A1 (en) | 2024-04-04 |
| CN115452929A (en) | 2022-12-09 |
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